Bacillus thuringiensis ("B.t.") is a gram-positive bacterium that typically produces proteinaceous crystalline inclusions during sporulation. These B.t. crystal proteins are highly toxic to certain insects. Crystal proteins from various B.t. strain isolates have been identified as having insecticidal activity against insect larvae from the insect orders Lepidoptera (caterpillars), Coleoptera (beetles), Diptera (mosquitoes, flies), and Homoptera (aphids). The insecticidal crystal proteins (ICPs) of B. thuringiensis were originally classified as CryI, CryII, CryIII, and CryIV proteins based on their insecticidal activities (Hofte H. and H. R. Whiteley, Microbiol. Rev. 53:242-255 (1989)). The Hofte and Whiteley (1989) review provides an overview of the B.t. insecticidal crystal protein genes. CryI proteins, which encompass crystal proteins of approximately 130-140 kilodaltons (kDa) in molecular mass, display lepidopteran toxicity. CryII proteins are approximately 71 kDa in mass and may display both lepidopteran and dipteran toxicity. CryIII proteins are approximately 73-74 kDa in mass and display coleopteran toxicity. The CryIV proteins represent a diverse group of proteins that exhibit dipteran toxicity. For the purpose of the present invention, the Hofte and Whiteley nomenclature will be used.
Commercial B.t. bioinsecticide products currently being marketed for lepidopteran insect control are based on either naturally occurring ("native") strains or transconjugants strains. Transconjugant strains are created by transferring a crystal protein-encoding plasmid from a donor strain to a recipient strain via a conjugation-like process, resulting in a new B.t. strain. Plasmids may also be transferred from one strain to another by phage transduction. Native and transconjugant strains are fermented in a broth medium, the spores and crystals harvested, either by spray-drying or by centrifugation, and subsequently formulated for spray application.
The insecticidal activity of conventional B.t. bioinsecticides results from insect larvae feeding on the crystal protein, typically in sprayed-on deposits of the bioinsecticide on leaves or other plant surfaces. General details of the mode of action of the insecticidal crystal proteins (ICPs) are apparent. The ICPs contained within the proteinaceous crystals are released into the insect midgut after ingestion and solubilization of the crystals. In many instances, the full-length proteins or protoxins are processed by midgut proteases to a fully active state. The 130-140 kDa CryI protoxins, for instance, are processed to a core toxin moiety of 60-65 kDa, derived from the amino terminal half of the full-length protoxin. This processed core toxin is regarded as the active toxin within the insect midgut. The discarded carboxyl domain of the protoxin, though not essential for toxicity, is apparently indispensable for CryI crystal formation in B.t. For purposes of the present invention, the carboxyl half of the CryI protein not contained within the active or core toxin will be referred to as the tail domain. As depicted in FIG. 1, the processed, activated toxin, derived from the chimeric CryIF-IAc crystal protein of the present invention, binds to the brush border membranes (BBMs) of the insect midgut epithelium, a step that frequently requires the presence of fortuitous "receptor" proteins. This binding is followed by an apparent intercalation event in which the active toxin moiety, or a portion of it, contributes to the formation of ion channels as well as aggregates to form larger pores within the BBM, leading to osmotic imbalance, cellular swelling and lysis. Intoxicated insect larvae stop feeding within minutes and eventually die.
For many lepidopteran insect pests, such as armyworms, the B.t. spore present in the bioinsecticide formulation also contributes substantially to toxicity. The synergistic effect of spores has been reported for a number of important lepidopteran insect pests, including Spodoptera exigua (Moar, W. J., et al., Appl. Environ. Microbiol. 61:2086-2092 (1995)), Lymantria dispar (DuBois, N. and D. H. Dean., Biological Control 24:1741-1747 (1995)), and Plutella xylostella (Tang, J. D., et al., Appl. Environ. Microbiol. 62:564-569 (1996)). This spore effect on the insecticidal activity of B.t. is apparently due to septicemia: the ability of the spore to germinate within the insect midgut, to penetrate the disrupted midgut epithelium, and to enter and proliferate within the hoemcoel. For many lepidopteran insect pests, it is therefore desirable that the B.t. bioinsecticide formulation contain a mixture of spores and crystals to achieve maximal efficacy.
Among the lepidopteran insect pests, armyworms are particularly difficult to control, regardless of the insecticide used. Thus, there is a need for bioinsecticide products for armyworm control that are both efficacious and cost-effective. To satisfy this need, the amount of crystal protein produced by B.t. in fermentation should be maximized as much as possible in order to provide for its economic and efficient utilization in the field. Increased concentration of crystal protein in the formulated bioinsecticide promotes use of reduced amounts of bioinsecticide per unit area of treated crop, without reducing the actual amount of crystal protein applied per unit area, thereby allowing for more cost-effective use of the bioinsecticide product. Alternatively, increased fermentation yields of crystal protein, resulting in more concentrated formulations, may be used to increase the amount of crystal protein applied per unit area, thereby enhancing the performance of the bioinsecticide product.
Previous efforts to create mutants or variants of B.t. strains that show enhanced production of crystal proteins have related primarily to the production of coleopteran- or dipteran- toxic crystal proteins, not CryI lepidopteran-toxic crystal proteins. Also, most of these examples describe oligosporogenous or asporogenous (produce few, if any, spores) variants of B.t. that show increased crystal protein production. As noted above, the full production of spores is a desirable feature for a lepidopteran-active B.t. strain used for the production of a comnmercial bioinsecticide.
U.S. Pat. No. 5,006,336, issued to Payne, describes a native B.t. isolate (PS122D3), active against coleopteran insects, which produces more coleopteran-toxic protein (CryIIIA) than does an unrelated coleopteran-toxic B.t. strain, B.t. san diego. Strain PS122D3 is not a variant of B.t. strain san diego.
U.S. Pat. No. 4,996,156, issued to Zaehner et al., describes a dipteran-active B.t. israelensis mutant strain which produces crystal proteins but is asporogenous.
Published European Patent Application Publication No. O 099 30 of Fitz-James, describes mutants of B.t. israelensis, obtained using a chemical mutagen, that produces up to 1.5 times the amount of dipteran-toxic crystal protein as does the progenitor strain.
Published European Patent Application Publication No. O 228 228, of Mycogen Corporation, describes asporogenous Bacillus thuringiensis mutants obtained by treatment of the progenitor strains with ethidium bromide. Such B.t. mutants are described as being more efficient at producing coleopteran-toxic (CryIIIA) crystal protein.
Published PCT International Patent Application Publication No. WO 91/07481, of Novo Nordisk A/S, describes a mutant of Bacillus thuringiensis tenebrionis, which was obtained by gamma irradiation and which produces two times the amount of coleopteran-toxic crystal protein (CryIIIA) obtained from the progenitor strain.
U.S. Pat. No. 4,990,332, issued to Payne et al., describes a lepidopteran-toxic B.t. kurstaki mutant strain (PS85al-168) that produces crystal protein in amounts "equal to or higher than the wild type" but is asporogenous.
The efficacy and cost effectiveness of a B. thuringiensis-based bioinsecticide product may also be improved by manipulation of the crystal protein composition in the bioinsecticide product, engineering of crystal proteins for improved insecticidal activity or stability, and by improvements in the formulation that promote longer shelf-life and longer persistence upon field application.
With respect to engineered crystal proteins, Geiser and Moser, in published Canadian Patent Application No. 2035199, disclose a chirneric CryIAb-CryIAc protoxin that shows improved temperature stability when compared to the native CryIAb protoxin. The native CryIAb protoxin does not form crystal protein inclusions in B. thuringiensis when grown at temperatures of 30-35.degree. C., the preferred temperature for cultivation of B. thuringiensis, and therefore cannot be produced efficiently in B. thuringiensis under these conditions. The chimeric CryIAb-CryIAc-protein, in which most of the carboxyl half or tail domain of the CryIAb protein has been replaced with that of the CryIAc protein, forms crystal protein inclusions in B. thuringiensis at 30.degree. C. and thus can be produced efficiently. This chimeric protoxin was reported to have the same toxicity profile for H. virescens and Trichoplusia ni larvae as the parental CryIA(b) protoxin (Aronson, A. 1993. Insecticidal toxins in Bacillus subtilis and other gram-positive bacteria. Sonensheim, A. L., Hoch, J. A., and Losick, R. (eds.). Am. Soc. Microbiol., Washington D. C., pp. 953-963). Furthermore, the reciprocal chimeric protein, in which most of the carboxyl half or tail domain of the CryIAc protein has been replaced with that of CryIAb, does not form stable inclusions in B.t. at the preferred cultivation temperature thus mimicing the temperature sensitive crystal forming phenotype of CryIAb.
Thompson et al., in PCT International Publication No. WO 95/30752, disclose a chimeric CryIC-CryIAb protoxin that shows improved insecticidal activity when produced in Pseudomonas fluorescens. This protoxin alone may not be produced efficiently in B. thutingiensis at the preferred cultivation temperature because it contains the tail domain of CryIAb that confers the temperature-sensitive crystal-forming phenotype of CryIAb described by Geiser and Moser.
A cryIF lepidopteran-toxic crystal protein gene is described in U.S. Pat. No. 5,188,960 issued to Payne et al., and in PCT International Publication No. WO 91/16434 of Ecogen Inc. Chambers et al. (1991) J. Bacteriol. 173:3966-3976, discloses a cryIF crystal protein gene from B.t. strain EG6346 subsp. aizawai and the insecticidal activity of its encoded CryIF crystal protein. A recombinant B.t. strain expressing this cryIF gene and designated strain EG7826 is disclosed by Baum in PCT International Publication No. WO 95/02058. Thompson and Schwab, in PCT International Publication No. WO 95/30753, disclose a chimeric CryIF-CryIAb protoxin that can be produced more efficiently in Pseudomonas fluorescens than the native CryIF protoxin. By analogy to the work of Geiser and Moser, the production of this chimeric CryIF-CryIAb protoxin in B. thuringiensis , at the preferred cultivation temperature, would be expected to be less efficient than that of the native CryIF protein. Thus, the requirements for efficient expression in B. thuringiensis and Pseudomonas appear to differ.
U.S. Pat. No. 5,508,264, issued to Bradfisch et al.discloses a synergistic effect as a result of the combination of the chimeric CryIF crystal protein and a chimeric CryIAc crystal protein produced in Pseudomonas fluorescens. The disclosed chimeric CryIF proteins includes the chimeric CryIF-CryIAb protoxin or a chimeric CryIF-CryIAc/CryIAb protoxin. The protoxin domain of the latter protein is comprised of both CryIAb and CryIAc sequences.
There is a need to improve the efficacy and cost-effectiveness of lepidopteran-toxic insecticides, particularly those used for the control of armyworms. This may be achieved, in part, by improving the amount of CryI crystal protein obtained in fermentation, thereby allowing for more economic use of the crystal protein in bioinsecticide formulations. The toxicity of the bioinsecticide product towards armyworms may be further improved by the use of a Bacillus thuringiensis strain that is proficient in the production of spores as well as in the production of CryI crystal protein. As evidenced by the prior art, standard methods for optimizing the production of CryI crystal proteins in a spore-forming entomopathogenic bacterium such as Bacillus thuringiensis remain elusive.